Method and apparatus for sorting biological cells with a MEMS device

ABSTRACT

A micromechanical actuator for sorting hematopoietic stem cells for use in cancer therapies. The actuator operates by diverting cells into one of a number of possible pathways fabricated in the fabrication substrate of the micromechanical actuator, when fluorescence is detected emanating from the cells. The fluorescence results from irradiating the cells with laser light, which excites a fluorescent tag attached to the cell. The micromechanical actuator thereby sorts the cells individually, with an operation rate of 3.3 kHz, however with the massively parallel 1024-fold device described herein, a throughput of 3.3 million events/second is achievable.

CROSS REFERENCE TO RELATED APPLICATIONS

This U.S. patent application is a continuation-in-part application ofU.S. application Ser. No. 10/189,607, filed Jul. 7, 2002.

FIELD OF THE INVENTION

This invention relates to the sorting of biological cells. Moreparticularly, this invention relates to the use of a MEMS device forperforming the sorting by physically separating the component ofinterest from the rest of the fluid sample.

BACKGROUND OF THE INVENTION

Many new therapies for cancer patients relate to enabling them to betterwithstand the challenge made to their bodies by the chemotherapies. Inparticular, it has recently been found that the inability of somepatients to cope with chemotherapies has to do with the destruction ofhematopoietic stem cells (HSCs), as ancillary damage of thechemotherapy. HSCs are the progenitor cells found in bone marrow,peripheral blood and many lymphoid organs. HSCs are responsible forgenerating the immune system components, such as T-cells, as well as thevital components of blood. When HSCs are destroyed in sufficientnumbers, it becomes difficult for patients to replace blood cells,resulting in anemia often suffered by patients. The destruction of HSC'sis also a leading cause of death in radiation victims, as the progenitorcells are destroyed, thereby destroying the ability to regenerate thevital components of the blood and immune systems.

Recent research has indicated however that if the HSCs are removed fromthe patients' bodies prior to their receiving chemotherapy, and thenreplaced after the chemotherapy, the HSCs are shielded from the effectsof the chemotherapy. By reinfusing the HSCs after the chemotherapy isfinished, the patients' ability to regenerate their blood cells isregained and their resilience to the therapy is greatly enhanced. As aresult, higher dosages of the chemotherapy can be administered topatients with better chances of diminishing the viability of the cancercells, and yet the patients are able to regraft their blood-formingHSCs, which have been protected from exposure to the chemotherapy.

Until recently, the standard treatment for patients requiringblood-forming system reconstitution after chemotherapy was a bone marrowtransplant (BMT). Bone marrow transplants require up to 100 withdrawalsof marrow from the hip bone by large needles and the subsequentreinfusion of large volumes of cells and other fluid. These proceduresare highly invasive, cumbersome, expensive and pose additional risks tothe patient.

Mobilized peripheral blood (MPB), which accomplishes the samepost-chemotherapy reconstitution with less trauma to the donor, can begenerated in most patients by injecting a granulocyte colony-stimulatingfactor (G-CSF) that causes the body to produce a sufficient quantity ofhematopoietic stem cells (HSCs). These cells migrate from the bonemarrow to the blood, from which they are harvested in a sufficientquantity in a single 24 hour session that only requires vein access.

Both the bone marrow extractions and mobilized peripheral blood fromcancer patients contain the hematopoietic stem cells necessary forreconstitution; however, they also contain large numbers of cancercells, which are reinfused into the patient along with the HSCs afterthe chemotherapy treatment. Logic and an increasing body of literaturesuggest that this reintroduction of cancer cells is one cause of thelimited survival improvement associated with high dose chemotherapy andcell transplant.

Therefore, technology was developed to obtain highly purifiednon-cancerous HSCs from mobilized peripheral blood; i.e., thepurification process eliminates the cancer cells, but retains thehealthy stem cells necessary for reconstitution. The purificationprocess also reduces the transfusion volume to less than 0.1 ml, incontrast to the 500-1500 ml of cells in fluid volume for BMT and MPB.The purification process is performed by flow cytometry, which separatesthe constituents of a fluid sample mixture according to fluorescencedetected from the constituents. Purity of the resulting HSC product was95% by this method, with no detectable cancer cells, and further detailsof the methodology can be found in Negrin et al., “Transplantation ofHighly Purified CD34⁺Thy-1⁺ Hematopoietic Stem Cells in Patients withMetastatic Breast Cancer”, Biology of Blood and Marrow Transplantation6:262-271 (2000). For patients undergoing this HSC reinfusion treatment,the 5-year survival rate for women with advanced metastatic breastcancer jumped from 5% to about 50%.

Another application for HSC sorting is protection against nuclearradiation effects. The procedure would be to sort HSCs from individualswho potentially could be exposed at some later date to nuclearradiation. The HSCs are frozen and can survive in that state essentiallyforever. If the individual is exposed, as could be the case in a nuclearplant accident or warfare, the HSCs are then shipped to the patient'slocation, rapidly thawed, and then re-inserted into the patient. Thisprocedure has been shown to save animals exposed to otherwise lethaldoses of radiation.

However for these treatments to become practical, it must be learned howto sort large quantities of viable hematopoietic stem cells from theother constituents of the blood, with high concentration and highpurity. An estimate of the number of stem cells required is 4×10⁶ stemcells/kg body weight. The present separation process, flow cytometry,uses a high-pressure nozzle to separate tiny droplets containing thecells. The cell suspension is brought to the nozzle assembly underpositive pressure, and introduced to the center of the sheath flow. Theproperties of fluid laminar flow focus the cell suspension into a singlefile, which is confined to the center of the fluid jet. Droplets areformed as the fluid exits the nozzle, and the droplets pass through oneor more laser beams, which irradiate the cells and excite fluorescentmarkers with which the cells are tagged. The droplets are then given anelectric charge to separate the droplets containing HSCs from thosecontaining other constituents of the blood, as detected by fluorescenceof the tagged molecules. The droplets are separated by passing thembetween a pair of electrostatic plate capacitors, which deflect thecharged droplets into a sorting receptacle. The time-of-flight of thedroplet through these stages requires careful calibration so that thesorting efficiency and effectiveness can be optimized.

Among the difficulties with the process is speed, as throughputs arelimited to about 40,000 events per second. The rate is limited by theamount of pressure that the cells can withstand without damaging theirviability, and the flow rate is proportional to the pressure. Thefluidic settings which control the conditions of operation of the flowcytometers are interrelated. The nozzle diameter, system pressure anddroplet frequency are independently set, whereas the jet velocity isrelated to the system pressure and nozzle diameter. Therefore thedroplet time-of-flight must be set by empirical calibration with astandard sample. Therefore, not only are the systems themselves quiteexpensive, they require trained engineering staff to operateeffectively. And lastly, contamination of the vessels with old sampleissue is a problem, as the equipment is difficult to sterilize.Decontamination issues encourage the use of disposable vessels, forwhich these machines are presently not designed. The high pressures usedin the machines favor permanent fixturing of the plumbing in the tools.Also the careful alignment required of the receptacles with thetrajectories of the droplets favors the permanent installation of thereceptacles. About 7000 such systems exist worldwide today, and tend tobe research tools rather than production equipment which can be used forclinical sorting in treating patients.

SUMMARY OF THE INVENTION

Therefore, a need exists for a separation technique that solvesthroughput, cost, and disposability issues associated with presentmethods. This disclosure describes a novel cell sorting device andmethod based on microelectrical mechanical systems (MEMS). MEMS devicesare micron-sized structures which are made using photolithographicaltechniques pioneered in the semiconductor processing industry. Due totheir small size and the batch fabrication techniques used to make thestructures, they are capable of massive parallelism required for highthroughput. These same features make them relatively inexpensive tofabricate, so that a disposable system is a realistic target for design.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood more fully from the followingdetailed description, and from the accompanying drawings, which however,should not be taken to limit the invention to the specific embodimentsshown but are for explanation and understanding only.

FIG. 1 is a simplified side view of the MEMS cell sorter chip, showingthe light channel layer and reflective layers in detail.

FIG. 2 is a plan view of the top surface of the MEMS cell sorter chip,showing the optically transparent light channel layer, as seen throughthe optical cover.

FIG. 3 is a plan view of the actuator/manifold layer of the MEMS cellsorter chip, showing the sorting manifolds.

FIG. 4 is a simplified diagrammatic view of the MEMS cell sorter system.

FIG. 5 is an enlarged top-down view of the sorting apparatus, amicromechanical actuator.

DETAILED DESCRIPTION

The MEMS device is an array of 1024 vertical inlet channels fabricatedin a wafer, wherein the 25 um diameter of each channel is just largeenough to admit the passage of a hematopoietic stem cell. (Hematopoietcstem cells are typically between and 10 um in diameter.) At the exitfrom each vertical channel is an independent valve/actuator. Theactuator directs the cells individually into one of two of differentpossible pathways, which are microfluidic channels etched into thewafer, beneath the vertical inlet channels. The situation is shownschematically in FIG. 1. The figure shows the application of the deviceto the separation of the components of human blood, in this case theseparation of hematopoietic stem cells (HSCs) from a fluid mixture ofother cells. The actuator separates the sample stream into one of twomanifolds, depending on the detection of a laser-induced fluorescencesignal or multiple signals, depending on the fluorescent markers used.The presence of fluorescence or multiple fluorescence indicates that anHSC is detected, and the actuator directs the cell into a stem cellmanifold with its stem cell receptacle. The receptacle contains acushion of fresh serum for sustaining viability of the cells collected.

The use of fluorescent markers to tag biological cells is known in theart. A wide variety of fluorescent markers exist which may be conjugatedas labels to antibodies specific to cellular markers which identifyparticular sets and subsets of cells. Fluorescent markers which areavailable include fluorescein, quantum dots, Texas Red,phycobiliproteins, cyanine derivatives and rhodamine. For example,Negrin et al. (“Transplantation of Highly PurifiedCD34+Thy-1+Hematopoietic Stem Cells in Patients with Metastatic BreastCancer”, Biology of Blood and Marrow Transplantation 6:262-271 (2000))reported that simultaneous detection of antigens CD34 and Thy-1 havegood correlation to the presence of human HSCs. The lack of fluorescenceindicates the cell is another constituent of the mixture, and not thetagged component. The occurrence of fluorescence indicates that thecomponent of interest is present. In the case of detection of multiplefluorescent markers simultaneously, in some cases more than one lasermay be used to excite the markers.

The sample cells are dispersed in any convenient medium which canmaintain viability such as phosphate-buffered saline, containing 0.1% to0.5% fetal calf serum. The cells may have been subjected topre-treatment, such as removal of cells by centrifugation, affinityseparation or other technique which provides enrichment of thepopulation of cells of interest. In addition, the cells may be dilutedto avoid cells being concentrated too close to each other. The fluidmixture is then introduced to the MEMS device under positive pressure,through the inlet via 96, and out through the outlet via 98. Thepositive pressure is chosen to select the proper flow rate through theMEMS chip, and can be set and fixed for the duration of the use of thechip. The MEMS chip wafer includes an optical cover 80 which is abarrier to the fluid mixture as well as an optically transparent elementwhich allows the fluorescent signals to leave the chip and be detectedoutside the chip. Spacer layer 82 separates optical cover 80 from lightchannel 88, and defines the thickness of the channel through which thefluid mixture flows before it enters the laser light/blood cellinteraction zone and vertical flow channel, 108.

As the fluid mixture enters through the inlet via, it floods the void 83which lies between the optical cover 80 and the light channel 88. Thelight channel 88 is made of glass (typically SiO₂) as shown in FIG. 1.Light channel 88 is sandwiched between two reflecting layers, lightreflecting layers 198 and 199. The function of the light channel is toguide laser light in a quasi-two-dimensional sheet, exposing the cellsin the fluid mixture only as the cells fall into the vertical channels108. The fluid mixture flows from the void 83 into the 1024 verticalchannels. The vertical channels have been formed in the light channel 88by lithographic patterning and etching, and provide a region 108 forinteracting with the laser light before the fluid mixture enters valvelayer 86. In region 108, the cells interact with the laser beam, and thecells of interest, which have been appropriately tagged with fluorescentmarkers, fluoresce as a result. The fluorescence is detected outside theMEMS chip and the fluorescing cell is mechanically separated from theother cells in the mixture, by the action of the electrostatic MEMSactuator. The valve labeled 90 is in the sort/save positioncorresponding to the presence of an HSC, whereas the valves labeled 92are in the waste position.

The top view of light channel 88 is shown in detail in FIG. 2, includingthe vertical channels, 108. The sample mixture is delivered to the topsurface of FIG. 2 by the inlet via, 96, from which it filters downthrough the vertical channels 108 to the actuator/manifold layer. Themanifold and actuator layer is shown in FIG. 3, and it lies just beneaththe optically transparent layer. The actuators are showndiagrammatically as the plurality of structures 92, lying at the exit ofeach vertical channel. As in FIG. 1, each of the actuators 92 shown isin the “waste” or “return” positions, directing the cells into the bloodreturn manifold 100, with the exception of actuator 90, which is in thesort/save position. This actuator directs a fluorescing cell into thestem cell manifold 94, and the remaining actuators 92 directnon-fluorescing cells into the blood return manifold 100. After beingproperly herded into the stem cell manifold, the cell follows the fluidstream under positive pressure, until it reaches the stem cell out tube104 leading to the stem cell receptacle, or the waste out tube 106leading to the waste receptacle if it is a non-fluorescing cell. Thedual manifolds have been patterned in the MEMS substrate, bylithographic means, as is shown in FIG. 3. The manifolds are sealed atthe top by eutectic layer 99, which lies between bottom light reflectinglayer 199 and the MEMS valves layer 86.

The timing of fluorescence detection, actuation and actuation back tothe nominal position 92 is important so as to allow only the fluorescingcell to be sorted and minimize the chance that an errant,non-fluorescing cell be sorted mistakenly. In the nominal case, the flowrate through each channel is roughly 0.2 meter per second. Just beforeor as the cell enters vertical channel 108, it begins fluorescing. Thethickness of light channel 88 is chosen to be roughly 30 um, so that thecell is lit by the laser light for 150 us. The fluorescing light isdetected in the first 100-200 us, and the actuator is immediately (withsmall computer/controller delay of only tens of microseconds) moved intoposition shown as sort position 90. This actuation takes approximately100 us. Therefore, the actuator is in the sort position just as the cellis approaching the MEMS valve layer 86. MEMS valve layer 86 is alsoapproximately 30 um thick. After actuation from position 92 to 90, theactuator pauses in the sort position for only 100 us, and is thenactuated back to position 92. While the actuator can move back withoutactive actuation, simply from relaxation of the spring force, thisembodiment uses active retraction to increase speed.

The sort rate of the device is determined by the actuator response time,which is approximately 3.3 kHz. The total time of 300 microsecondsincludes 100 usec for moving the actuator to the sort position afterfluorescence is detected, a 100 usec pause to allow passage of the HSCinto the stem cell manifold, and 100 usec for moving the actuator backto the non-sort position. Considering the 1024 such channels, and avelocity of flow in the constricted channels described herein of 0.2m/sec, the device will pass 1 liter of fluid mixture in 2 hours. The 3.3kHz sample rate translates into an overall throughput of 3.3 millionevents/sec, taking into account the 1024 parallel channels. Thisthroughput is almost two orders of magnitude better than the fastestflow cytometers. Typically, the cells are dilute in the fluid mixture,and the number of cells per second will depend on the dilution.

In order to maximize the flow of the fluid mixture without excessivepressures, the MEMS chip utilizes a large number of parallel channelsflowing through the plane of the wafer as well as across the plane ofthe wafer. The large number of short path, parallel channels through thewafer has the advantage that very large pressure gradients are notneeded to obtain reasonable flow rates. The device is designed so thatthe dominant pressure drop is generated in the vertical channel/actuatorregion only, and care is taken to provide a uniform pressure headpreceding the vertical channels and a minimum back pressure after theactuator region as the flow opens up into the larger manifolds. Thedevice also does not need to create or manipulate a fine spray ofdroplets; instead the flow is continuous. With the actuator acting as alow inertia knife-edge gate valve, relatively low forces are needed toperform the sorting. This keeps the sample rate high with the reasonablevoltages applied, on the order of 50 V. The tool is designed to be a lowcost, special purpose machine sorting into two buckets only, but theconcept is extendable to other applications.

The details of the optical system are shown in FIG. 4. Two lasers areused to allow the flexibility to induce fluorescence in multiplemarkers: i.e. a first Ar⁺ laser operating at 488 nm, and the second aRhodamine 6-G dye laser operating at 590 nm. The beams are combined witha beamsplitter/turning mirror 252, and focused into a line on the lightchannel 88 by a cylindrical lens 256. The two dimensional sheet of lightpropagates within the light channel 88. Fluorescent light emanating fromvertical channels 108 (if an appropriate fluorescing cell is present)passes out of the MEMS chip through optical cover 80 and the lens 260directs an image of the chip surface onto the set of CCD cameras 264 and265, through the set of filters 266 and 267. The filters are used toselect only the desired fluorescence signal of the marker. In the caseof sorting HSCs in which both CD34 and Thy-1 antigens are used, thefilters are selected to pass only the wavelengths for the tags for thoseantigens, respectively. In general, then, the camera detectors are inthe dark except during the rare events of detection of a fluorescencesignal. The detection of fluorescence by the CCD array (or thesimultaneous detection of both signals, one in each camera) indicatesthe presence of an HSC in the sample manifold, at the position in thearray indicated by the CCD camera. The electronics then causes theappropriate actuator to be energized, diverting the sample cell into theappropriate manifold. The actuator is then positioned back to itsinitial state.

In the implementation preferred herein, the fluorescent light passingthrough lens 260 impinges first on one filter, filter 266. Light of theproper wavelength passes through filter 266 into the first high speedvideo camera 264. All other light reflects from the surface of filter266, and impinges on filter 267. Light of the proper wavelength for thatfilter passes through into the second high speed video camera 265. Inthis way, efficient use is made of the available light to optimizesignal-to-noise and speed in the system.

A variety of state-of-the-art camera systems are available to serve asthe high-speed cameras. For example, Photron USA (San Diego, CA) marketsthe PhotoCAM 250 CL, a monochrome camera with 10,000 frames per secondperformance (adequate for the 100 microsecond requirement in thisinvention) with over 4000 pixels in each frame, sufficient for thisapplication. Although this high speed camera is not as sensitive asphoto-multiplier tubes commonly used in modem cell-sorters, gain comesfrom the longer integration time in the current invention, ten timeslonger than the cell sorters, so that adequate signal-to-noise isachieved using cameras. If additional sensitivity is required for aparticular application, an intensifier plate can be added in front ofthe camera's detector. These are common in industry, known asmicrochannel plates (MCP), and are an array of channeltrons.

In practice, filters 266 and 267 may not be individual filters, butfilters on respective filter wheels, so that one particular filter canbe selected simply by rotating the wheel. In this way, the machine caneasily be configured to detect different wavelengths.

General-purpose computer 268 directs the operation of the variouselectronics units through the 2060-pin connector (2048 lines plus 12ground lines) 260 to control the actuators, and CCD harness 262 toacquire the signal from each camera detector. The general purpose PCalso controls laser pulse timing, if a pulsed laser is used. The bloodis delivered to the chip and the waste and sorted cells are taken awayfrom the chip through the set of plumbing tubes, 270, typically made ofstainless steel, and glued into the MEMS chip.

The action of the micromechanical actuator can be understood byconsidering the details of the actuator as seen in FIG. 5. Theelectrodes are formed in comb shapes 362 and 364 with interlacingfingers on the actuator 360, to increase the surface area and thereforethe torque at a given voltage. As shown in FIG. 5, there are three viasand electrical connections to the MEMS valve layer. One via connection352 is made to comb 362, one 354 is made to comb 364, and the third 350connects to the moving actuator interlacing fingers 360 as well as tothe rest of the silicon layer which is not the interlacing fingers, andcan be thought of as the ground of the device. There are twelve groundconnections that are brought through the substrate, making the totalnumber of pins out equal to 1024+1024+12=2060. In this way, the fluidmixture is not exposed directly to electric fields; rather the fieldsare isolated between the comb area and the interlacing fingers of theactuator. Sub-micron lithography yields very sharp features, with highsurface energy corners and gaps of less than 2 um, too small to allow aHSC flowing in from the vertical channel 108, to leak between the combs.The valve is normally in the position to direct flow into the wastemanifold 374, but can be actuated within 100 us to redirect the flowinto the stem cell manifold 376.

A requirement of the actuator is that it have sufficient force towithstand the pressure of the fluid mixture and hold its properposition, directing flow into the appropriate manifold. Straightforwardfluid calculations show that forces on the order of 10e-9 N arerequired, and these are easily attained with the current electrostaticactuator configuration. The action can be seen in the top-down viewshown in the figure, with the sample cell entering the actuator areafrom the top through the vertical inlet 108, and flowing downward intothe waste receptacle, as the actuator blocks the upward path leading tothe stem cell manifold. In the actuated state however, the actuator 360is drawn down by electrostatic attraction to the lower comb 362, closingoff the lower route to the waste receptacle and opening the upper routeto the stem cell manifold. The hinge area of the actuator is designed tohave the restoring force required to return the actuator to its startingposition at the upper comb, and separated from the lower comb, closingthe stem cell path and opening the path to the waste manifold andreceptacle. In our preferred embodiment, however, we include a secondupper comb electrode, 364, to retract the actuator to its originalposition. When a voltage is applied to upper comb 364, the valve isretracted within approximately 100 us to the normal position, whichdirects the flow once again to the waste manifold 374.

It should be pointed out that at all times, independent of the positionof each valve/actuator, the saline solution which makes up the bulk ofthe fluid mixture is flowing into both the waste manifold 374 as well asthe stem cell manifold 376. This is because of the approximate 1 microngaps which exist between the valve wafer and the light channel 88,between the actuator gate and the sidewall of the manifold, and betweenthe actuator gate and the silicon substrate 94. This continuous flowdoes not act to the detriment of the device, however, since the gaps aremuch smaller than the size of the cells to be sorted. This is evidentfor the case of HSCs (5 to 10 um in diameter and roughly round). If thecells to be sorted are very small, nearing 1 um, then greater care wouldhave to be taken to decrease the gaps. For example, 0.3 um resolutionlithography is readily available, although not widely used in MEMStechnology, and can be applied to reduce the gaps involved here, as wellas reduced thickness wafer bonding lines and insulator thickness on thesilicon-on-insulator (SOI) wafer which comprises the actuator wafer.

To discourage wetting of the wafer surfaces, the actuator valve wafer iscoated with a thin fluorocarbon film, approximately 10 to 20 Angstromsthick, and with some bonding affinity for the wafer surface. Examples ofthe fluorocarbon material include AM2001 or Z-Dol, common lubricantssold by Dupont Corp. (Wilmington, Del.). Coating with such films iscommon in industry, for example it is used on thin-film disks fordisk-drive storage. The function of the fluorocarbon film is to reducewetting of the wafer surfaces. While the fluid mixture is driven bypressure through vertical channels 108, past the valve and into one ofthe manifolds 376 or 374, the fluid mixture will not wet or flow easilyinto the comb region defined by the actuator 360 and the upper and lowercombs 362 and 364. Fluid mixture in this region is not desired, as thefluid is conductive as well as being viscous. The conductivity willinterfere with the resulting electrostatic force generated by applying avoltage to the interlacing fingers, and the viscosity will slow down theactuator speed. In the event that there is too much leakage of the fluidmixture into the interlacing finger area and the device function suffersfor a particular application, pressurized gas (air, nitrogen, forexample) can be provided to the MEMS chip through a via in the bottom ofthe chip, with slight overpressure to keep the interlacing region dry.

The blood enters the MEMS chip from a regulated pressure supply through2 filters, a first small pass filter at 20 um, and a subsequent largepass filter at 3 um. The filters assure the particles are within theexpected size range, and filters out debris which could otherwise clogthe small passageways. The output of the filters feeds the MEMS sortingchip. The plumbing lines, which contain the flow, are bonded permanentlyto the chip, making the package entirely disposable. Sterilization istherefore not an issue.

The MEMS chip is created by standard processing now common in the MEMSindustry. The MEMS chip is made by making two separate wafers, theactuator wafer and the optical wafer, bonding these wafers together, andsubsequently dicing the wafer into constituent dies, which are the MEMSchips. For example, for a 6″ diameter wafer, each wafer comprisesapproximately 40 to 50 MEMS chips.

The optical wafer includes optical cover 80, spacer 82, void 83, andlight channel 88, as shown in FIG. 1, and is made as follows. The waferstarts as a 640 micron-thick, 6″ diameter, silicon dioxide (glass)wafer, widely available. As the optical wavelengths considered hereinare within the visible range, normal fused quartz glass wafers willsuffice. If the wavelengths demanded transmission in the ultraviolet,for example, single crystal SiO₂ could be used. Spacer 82 is then formedby depositing the spacer material (e.g. sputtered SiO₂, silicon nitride,plated NiFe), typically 30 to 40 microns thick, patterninglithographically, and then etching the spacer material where it is notrequired. The region, which will become void 83 is then made by platinga sacrificial material (such as copper) into the void 83 region. Theplating is performed much thicker than is required to fill up the void83 space, and then the wafer is lapped flat with chemical mechanicalpolishing, using spacer 82 as a lapping stop. The sacrificial materialis chosen in this case to lap much faster than spacer 82, and thus thewafer is made flat with spacer 82 and the sacrificial material fillingvoid 83 exposed. Light reflecting layer 198 is then formed by depositinga material such as chromium, gold, or titanium, (chromium is preferredin this embodiment) and the layer is approximately 1000 Angstroms thick.The optically transparent layer, light channel 88 is then deposited, andcould be either SiO₂ or Al₂O₃, with sputtered SiO₂ preferred in thisembodiment, approximately 30 microns thick. Then light reflecting layer199 is deposited, again 1000 Angstroms of chromium in the preferredembodiment.

One of the two materials making up the eutectic wafer-bonding material,eutectic 99, is then deposited and patterned. For example, the preferredmaterial is a gold-indium eutectic, and so 1000 Angstroms of gold isdeposited everywhere. The optical wafer is then patterned and etched toremove the gold, chrome, glass and other chrome layer in the region ofvertical channels 108 and the inlet via 96. The patterning is standardphotolithography, and the etching can be done either with wet etchantsor reactive ion etching (RIE). The preferred embodiment herein is RIE,as the wall angles are very controllable and the chemistries are wellknown and practiced. The wall angle is chosen to be slightly offvertical, with the narrow section at the top of the vertical channels108 to avoid the possibility of a particle becoming trapped inside thechannel. Lastly, the optical wafer is completed by etching away thesacrificial material, leaving void 83 empty. In the preferredembodiment, the sacrificial material is copper, and it can be removed byexposure to a liquid etchant.

The actuator wafer may be built starting with an SOI wafer(silicon-on-insulator). The starting material may be 640 micron-thicksilicon, highly resistive in our preferred embodiment using very lowdoping such as float-zone silicon, with a 1 micron SiO₂ layer sandwichedbetween the thick silicon and an “active layer” silicon wafer. Theactive layer may be chosen to be approximately 30 microns thick in thiscase. The electrical vias may be formed first, by patterning the back ofthe actuator wafer, using deep reactive ion etching (DRIE) to etchvertical walls in the silicon all the way to the SiO₂ etch stop. TheSiO₂ is removed at the bottom of the vias using RIE or wet chemistry(brief hydro-fluoric acid dip), a plating-base is sputtered or depositedby ion-beam deposition (1000 Angstroms of copper) and copper is platedinto the holes, filling them beyond the surface of the actuator wafer.The surface is then lapped with chemical mechanical polishing untilflat, leaving the copper flush with the wafer surface backside.

Blood In, Blood Return, and Cell Sorting channels are also etched intothe actuator wafer substrate using patterning and DRIE etching.

The front side of the actuator wafer, which is the “active layer”silicon of 30 microns thickness, is then patterned and etched with DRIE,forming the actuator, interlacing combs, inlet via, stem cell manifoldand blood return manifold with one etch. The etch stop is the 1micron-thick SiO₂ layer of the original SOI sandwich. The othercomponent of the eutectic is then applied by patterning and plating 1micron of indium in this embodiment. The pattern is such that the indiummakes a seal against the optical wafer in all areas except the movingactuator gate. In that area, there is no indium, and so the indiumprovides both one component of the eutectic seal as well as the thinstandoff allowing actuator movement. The eutectic bond line between theoptical wafer and actuator wafer also serves as the grounding connectionbetween the 1024 actuators. The SiO₂ layer is then etched to free theactuator, preferably using a brief hydro-fluoric acid dip, well knownthe MEMS art. The actuator wafer is then dipped in a highly-dilutefluorocarbon mixture (AM2001 from Dupont Corporation is the preferredembodiment) to apply a 15 Angstrom layer of the lubricant over thesurface.

The optical and actuator wafers are then bonded together using a WaferBonder, typically a Karluss (Waterbury Center, Vt.) aligner/bonder, inwhich the temperature is chosen to enable the two materials chosen toform a eutectic bond. In the case of indium and gold used here, atemperature of 180 degrees centigrade will suffice.

On the bottom side of the actuator wafer, standard bumping processes onthe exposed copper vias can be used to create a ball grid array forsurface or board mount applications. Therefore the design has theadvantage of bonding the transmission lines of the device directly tothe input lines, avoiding the impedance mismatch associated with wirebonding. The vias can also be used for wire bonding pads for situationswhere surface mount technology cannot be used.

Setup and operation of the MEMS chip and system described is as follows:the MEMS chip is placed onto the pin array structure with pin harness. Asample fluid mixture of saline solution and dilute 5 um beads with theproper attached fluorescent markers for the anticipated cell sorting isthen applied to the chip. The lasers irradiate the fluid as it entersthe vertical channels, and the resulting fluorescence is collected bythe lens and imaged onto the CCD cameras. The computer then integratesthe output of the video signal and identifies the exact position of thevertical channels 108, as imaged by the lens filter/camera. In this way,the system can adapt to some variability in the exact alignment of thedisposable chip in its receptor. After the sample fluid is flushed fromthe chip, the system is ready to sort cells.

The invention described herein has several features, which enhance itsreliability. In order to sort biological cells and contemplate treatmentfor cancers, etc., the system must be considered to be very safe insorting, that is the charices of mistakenly sorting a cancer cell mustbe very low. The fail-safe position of each actuator/valve for the MEMSchip is to not sort the cell. If any actuator fails to function, itconstitutes only a slight loss in efficiency of sorting (for example,one non-functioning actuator represents only 0.1% loss in efficiency inthe current configuration), but does not pose a risk to the patientconstituted by sorting the wrong cell. Similarly, the software runningin the general-purpose computer has fail-safe features. For example, ifthe fluorescent signal from a single vertical channel 108 remains on formore than approximately 400 microseconds, then that actuator isdisabled, since that particular channel may have become dogged, and onecannot take the risk of allowing an improper cell to be sortedmistakenly.

While the invention has been particularly described and illustrated withreference to a preferred embodiment, it will be understood by thoseskilled in the art that changes in the description and illustrations maybe made with respect to form and detail without departing from thespirit and scope of the invention. For example the electrostatic forcesmay be manipulated by choice of film thicknesses, hinge design andmaterial composition. Alternatively, electromagnetic forces,piezo-electric forces or thermal expansion forces could be substitutedfor electrostatic forces. Spring constants may be varied by changing theaspect ratio of the beams or stiffnesses of the hinges. The devicedescribed can be used to sort any molecule, which has been tagged withan appropriate fluorescent marker. Accordingly, the present invention isto be considered as encompassing all modifications and variations comingwithin the scope defined by the following claims.

1. A micromechanical actuator, comprising: a fabrication substrate; adriven member hingedly mounted to said substrate by one or more springs;an optically transparent layer covering the hingedly mounted member butallowing clearance for its movement, and further containing a channeldirecting the flow of a fluid mixture to the distal end of the hingedlymounted driven member; and a means for actuating said hingedly mountedmember in order to direct the fluid mixture along a path chosen from anumber of different microfluidic paths formed in said fabricationsubstrate.
 2. The micromechanical actuator of claim 1, wherein thechoice of which microfluidic path along which to direct the fluidmixture is based on fluorescence detected from a component of interestin the fluid.
 3. The micromechanical actuator of claim 2, wherein thefluorescence is induced by one or more lasers irradiating the fluid. 4.The micromechanical actuator of claim 2, wherein the fluorescence isproduced by a fluorescent marker previously attached to the component ofinterest in the fluid mixture.
 5. The micromechanical actuator of claim4, wherein the component of interest in the fluid mixture includesbiologically active cells.
 6. The micromechanical actuator of claim 4,wherein the component of interest in the fluid mixture includes humanhematopoietic stem cells.
 7. The micromechanical actuator of claim 4,wherein the component of interest in the fluid mixture includes a viruscell population.
 8. The micromechanical actuator of claim 1, wherein theactuation means is electrostatic.
 9. The micromechanical actuator ofclaim 8, wherein the hingedly mounted member is a first electrode with aset of conductive protuberances which interlace an opposing set ofprotuberances of a second stationary electrode, and is fastened in ahinged manner to one end of the second electrode, and the secondelectrode is energized to attract the first electrode away from itsinitial position, thereby directing the fluid mixture of cells into achosen path.
 10. The micromechanical actuator of claim 9, furthercomprising a third stationary electrode fabricated in the substrate, andenergized to retract the first electrode back to its initial position.11. The micromechanical actuator of claim 9, wherein the first hingedlymounted electrode is maintained at ground potential relative to thefabrication substrate, which is also maintained at ground potential. 12.The micromechanical actuator of claim 9, wherein the actuator isenergized by applying about 50V potential between the first hingedlymounted electrode and the second stationary electrode.
 13. Themicromechanical actuator of claim 10, wherein the first electrode isretracted to its initial position by application of about 50V potentialto the third stationary electrode.
 14. The micromechanical actuator ofclaim 10, wherein electrical access to the actuator electrodes is gainedwith through holes formed in the fabrication substrate, and the throughholes are deposited with a conducting material.
 15. A system forseparating a component of interest from a fluid mixture, said componenthaving been distinguished from the rest of the fluid mixture by theattachment of a fluorescent marker, said system comprising: An inlet viafor delivering the fluid mixture to a surface of a fabricationsubstrate, said surface having been prepared with an array of channelsso dimensioned as to permit the passage of the component of interestfrom the fluid mixture; One or more lasers for irradiating the fluidmixture while in or just before the channels; A detector for detecting afluorescence signal from the irradiated fluid mixture; and One or moremicromechanical actuators and their associated microfluidic structuresformed in the fabrication substrate beneath the channels, wherein themicromechanical actuators direct the flow of the fluid mixture along oneof a number of possible pathways in the microfluidic structures, inresponse to the detected fluorescence signal.
 16. The system forseparating a component of interest from a fluid mixture of claim 15,wherein the component of interest is a biologically active cell.
 17. Thesystem for separating a component of interest from a fluid mixture ofclaim 15, wherein the component of interest is a human hematopoieticstem cell population subset.
 18. The system for separating a componentof interest from a fluid mixture of claim 15, wherein the component ofinterest is a virus cell population.
 19. The system for separating acomponent of interest from a fluid mixture of claim 15, wherein themicromechanical actuators are covered by a thin layer of fluorocarbonlubricant.
 20. The system for separating a component of interest from afluid mixture of claim 15, wherein the detector is a charge-coupleddevice (CCD).
 21. The system for separating a component of interest froma fluid mixture of claim 15, wherein a microchannel plate intensifier isplaced before the detector.
 22. The system for separating a component ofinterest from a fluid mixture of claim 15, wherein the micromechanicalactuator is electrostatic.
 23. The system for separating a component ofinterest from a fluid mixture of claim 15, further comprising one ormore light filters arranged in front of the detector.
 24. The system forseparating a component of interest from a fluid mixture of claim 15,further comprising one or more lenses to image the irradiated fluidmixture onto the detector.
 25. The system for separating a component ofinterest from a fluid mixture of claim 15, further comprising acylindrical lens to focus the laser light onto the fluid mixture whilein or just before the channels.
 26. The system for separating acomponent of interest from a fluid mixture of claim 15, wherein theirradiating lasers include an Ar⁺ laser operating at about 488 nm, andalso a dye laser operating at about 590 nm.
 27. A method for separatinga component of interest from a fluid mixture, comprising: Preparing thecomponent of interest by contacting it with a fluorescent labeledmolecule which binds with the component of interest; Delivering thefluid mixture to a surface of a fabrication substrate, said surfacehaving been prepared with an array of channels so dimensioned as topermit the passage of the component of interest from the fluid mixture;Irradiating the fluid mixture prepared with the fluorescent-labeledmolecule with one or more lasers while in or just before the channels;Detecting the resulting fluorescent signal with a detection device;Choosing one of a number of different fluid paths, based on thedetection of a fluorescent signal; and Directing the flow of the fluidmixture into the chosen path using one or more micromechanical actuatorsformed on the fabrication substrate adjacent the channels, in order tosort the component of interest from the rest of the fluid mixture. 28.The method for separating a component of interest from a fluid mixtureof claim 27, wherein the micromechanical actuator is electrostatic. 29.The method for separating a component of interest from a fluid mixtureof claim 27, wherein the component of interest is a human hematopoieticstem cell population subset.
 30. The method for separating a componentof interest from a fluid mixture of claim 27, further comprisingdetecting the resulting fluorescence with a charge-coupled device (CCD).31. The method for separating a component of interest from a fluidmixture of claim 27, further comprising using a cylindrical lens tofocus the laser light onto the fluid mixture while in or just before thechannels.
 32. The method for separating a component of interest from afluid mixture of claim 27, further comprising imaging the irradiatedmixture in the channels with a lens onto the detection device.
 33. Themethod for separating a component of interest from a fluid mixture ofclaim 27, further comprising blocking all light except for thefluorescent wavelength associated with the fluorescent labeled moleculefrom entering the detector with one or more optical filters.